The goal of this research is to identify the molecular components of human cytotoxic T-cells (CTL) that mediate the cytolysis of alloantigen bearing human tumor cells in vitro. Long-term HLA-A2 or B7 Class I specific human CTL lines sensitized by mixed lymphocyte culture and grown in the presence of human T-cell growth factor will be used as a model system for studies of the lytic mechanism. Mice or rats immunized with CTL lines, subcellular fractions and purified membrane antigens will be used to derive monoclonal antibodies from hybrid cell lines produced by cell fusion with mouse myeloma cell line. Hybrid cells will be screened for the production of antibodies that directly inhibit the cytolytic activity of Class I specific human CTL. Secondary screening methods used for cloning of hybrid lines are the indirect binding assays detecting cell surface antigens and immunoprecipitation. The monoclonal antibodies obtained will be used to characterize the biochemical properties of their respective antigens including molecular weight of polypeptide subunits, quantitative cell surface expression, isoelectricpoint, and carbohydrate content. This information will be used to develop a purification procedure using monoclonal antibody affinity chromatography. Several types of experiments will be conducted to determine whether the monoconal antibody inhibits the functional activity of CTL by trivial means or by specific steric hindrance of a functionally important molecule. Identification of the phase of the multistep CTL lytic reaction (recognition, activation or lysis) inhibited by the monoclonal antibody relative to the Mg++ and Ca++-dependent phases and the sites(s) of inhibition by monoclonal antibodies to other CTL surface antigens T8, T3 and the lymphocyte function associated antigens (LFA) will form the basis for defining a molecular pathway of the human CTL lytic mechanism. This study will help establish the framework for further detailed molecular biological studies of the CTL lytic mechanism and the molecular regulatory pathways that control this effector system. It is hoped that the reagents developed identifying functionally relevant T lymphocyte molecules can be used in diagnostic and therapeutic procedures to explore the participation of CTL in a variety of tissue destructive diseases and normal homeostatic mechanisms.